The goal of this proposal is to explore the molecular mechanism as to why oligodendrocytes (OGs) prefer necroptosis, a regulated necrotic cell death pathway, as the primary cell death mechanism and the contribution of this pathway to progressive demyelination, inflammation and neurodegeneration in animal models of multiple sclerosis (MS). MS, an inflammatory demyelinating disease of the central nervous system (CNS), is the most common chronic neurodegenerative disease for young adults during their most productive ages. While the immunological basis of MS has been studied extensively, we still know very little about the mechanism that leads to the degeneration of OGs, the myelin producing cells that play a critical role in the maintenance of activity and integrity of neuronal axons. Preventing the death of OGs might be able to inhibit demyelination and axonal degeneration, the major cause of irreversible neurological disability in patients with progressive MS. Activation of TNFR1 by TNF has recently been shown to mediate two alternative cell death pathways: caspase-dependent apoptosis and caspase-independent RIP1 kinase-dependent necroptosis (programmed necrosis). However, for most cell types analyzed so far, necroptosis is only activated when the activation of caspases is inhibited by chemical inhibitors or by genetic mutation. Interestingly, we found that OGs undergo necroptosis upon stimulation by TNF alone which can be effectively blocked by Nec-1 or by RIP3 deficiency. We have shown that 7-Cl-O-necrostatin-1 (7-Cl-O-Nec-1), a highly specific inhibitor of RIP1 kinase, protects against TNF-induced oligodendrocyte death in vitro and two mouse models of MS in vivo [cuprizone model and experimental autoimmune encephalomyelitis model (EAE)]. In addition, RIP3-/- mice are also resistant to cuprizone model and RIP3-/- OGs are protected against TNF. We propose to investigate as to why OGs prefer to use necroptosis as the primary cell death pathway and the role and mechanism of RIP1 kinase in mediating the death of OGs. Specific Aim 1 is to investigate the role and mechanism by which cellular metabolism and redox state control the sensitivity of OGs to TNF. This is to test the hypothesis that the cell- cell interaction regulated high metabolic activity in OGs provides a critical mechanism that controls redox state and the sensitivity of OGs to TNF mediated necroptosis. Specific Aim 2 is to investigate the role of S- nitrosylation in regulating the sensitivity of OGs to TNF induced cell death. This is to test the hypothesis that elevated nitrosylation stress in TNF stimulated OGs leads to the inhibition of caspases and sensitization of OGs to necroptosis. Specific Aim 3 is to examine the involvement of RIP1 kinase in mediating necroptosis of OGs in vivo and in vitro using RIP1 kinase dead knockin mutant mice. Our study may provide a strong rationale for the development of RIP1 kinase inhibitors as an OG protective strategy for the treatment of MS, and an orally available, highly specific and nontoxic RIP1 kinase inhibitor, 7-Cl-O-Nec-1, as a lead compound.